Treatment of b-cell lymphoma with microrna

ABSTRACT

The invention relates to microRNA-34a and related microRNAs for use in the treatment of B-cell lymphoma. Likewise it relates to microRNA-34a for use in the preparation of a medicament for the treatment of B-cell lymphoma, and for a method of treatment of B-cell lymphoma comprising administering microRNA-34a. These claims are based on the observation that microRNA-34a shows strong anti-proliferative effects when overexpressed in diffuse large B-cell lymphoma (gDLBCL) cell lines, or when delivered intratumorally or systemically in xenograft models of DLBCL.

FIELD OF THE INVENTION

The invention relates to the use of microRNA in the treatment of B-celllymphoma.

BACKGROUND ART

Low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphomas areextranodal B-cell lymphomas that arise in the context of chronic gastricinflammation induced by persistent Helicobacter pylori infection(Sagaert X. et al., Nat Rev Gastroenterol Hepatol 2010, 7(6):336-346).In its early stages, MALT lymphoma is an indolent and localized diseasethat can be treated by antibiotic eradication therapy targeting theunderlying infection. In line with the concept that gastric MALTlymphomas are antigen-driven tumors, the surface immunoglobulins of MALTlymphoma B-cells are clonal, somatically hypermutated, and haveundergone positive selection. MALT lymphoma tumor immunoglobulins (Igs)are polyreactive, i.e. they bind with similar affinity to variousunrelated self and foreign antigens, and show a biased use of Ig V_(H)gene segments previously linked to poly- and autoreactive antibodies(Craig V. J. et al., Blood 115:581-591 (2010)). Early MALT lymphomasfurther require T-cell help in the form of soluble T-helper cell-derivedsignals, most likely B-cell mitogenic cytokines such as IL-4 and IL-5.

Low grade MALT lymphomas may progress to more advance disease, eitherthrough the acquisition of one of three characteristic chromosomaltranslocations resulting in the constitutive activation of the NE-KBsignaling pathway, or through the histologically evident transformationto high-grade gastric diffuse large B-cell lymphoma (gDLBCL). High gradetransformation of Helicobacter-associated MALT lymphoma accounts for themajority of gDLBCL cases, whereas primary gDLBCL is rare. gDLBCL ischaracterized by antigen-independent growth, resistance to Helicobactereradication therapy and a number of genetic alterations that maycontribute to high grade transformation. In particular, TP53 mutations,Bcl6 overexpression and the aberrant DNA hypermethylation of tumorsuppressor genes have been shown to be associated with high gradetransformation. However, the precise molecular mechanisms underlying thetransition from low-grade MALT lymphoma to gDLBCL remain largelyunclear.

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs, whichmodulate the expression of their target genes at thepost-transcriptional level. Aberrant expression of specific miRNAs hasbeen associated with both solid and hematopoietic malignancies,including chronic lymphocytic leukemia, lung cancer and ovarian cancer.The majority of human miRNAs are located at fragile sites or cancerassociated genomic regions. For example, the frequent down-regulation ofthe fragile region encoding miR-15a (microRNA-15a) and miR-16-1 promotesCLL through dysregulation of the Bcl2 oncogene. The widespreadderegulation of the miRNA transcriptome appears to be a hallmark ofcancer and has been attributed to deletions, amplifications or mutationsof miRNA loci, epigenetic silencing or aberrant transcriptionalregulation of miRNA genes. Many studies have revealed the potential ofmiRNA expression profiles as diagnostic and prognostic markers ofcancers, which may be more useful than expression analysis ofprotein-coding genes for the classification and stratification of cancersubtypes (Zhang B. et al., Dev Biol 2007, 302(1):1-12).

MicroRNA profiling in Burkitt lymphoma demonstrated a verycharacteristic MYC induced microRNA expression. This indicates that MYCregulates Burkitt lymphoma cell fate in a direct mode at thetranscription level and indirectly at the translational level (RobertusJ. L. et al., Brit J Haematol 2010, 149:896-899). In chronic lymphocyticleukemia (CLL) patients with TP53 abnormalities exhibit downregulationof miR-34a, miR-29c and mirR-17-5p (Mraz M. et al., Leukemia 2009,23:1159-63).

SUMMARY OF THE INVENTION

The invention relates to microRNA, such as microRNA-34a, for use in thetreatment of B-cell lymphoma, in particular gastric B-cell lymphoma,non-gastric diffuse large B-cell lymphoma, extranodal diffuse largeB-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia.Likewise it relates to microRNA, such as microRNA-34a, for use in thepreparation of a medicament for the treatment of B-cell lymphoma, andfor a method of treatment of B-cell lymphoma comprising administeringmicroRNA, such as microRNA-34a.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. MicroRNA expression levels of miR-34a and let-7a in gastritis,low grade MALT lymphoma and gDLBCL.

Quantification of let-7a (A) and miR-34a (8) expression in gastritis(G), low grade MALT lymphoma (MALT-L) and gastric diffuse large B-celllymphoma (gDLBCL) samples by LNA real-time RT-PCR; absolute expressionwas normalized to U6 snRNA.

ns: not significant.

FIG. 2. Myc is over-expressed in gastric diffuse large 8-cell lymphoma(gDLBCL) and exhibits oncogenic properties in DLBCL cell lines in vitro.

(A, B) Myc expression was analyzed by immunohistochemistry on a tissuemicroarray comprising 37 gDLBCL and 39 low grade MALT lymphoma (MALT-L)cases. The fraction of MALT lymphoma and gDLBCL cases with high (black),low (grey) and no Myc expression (white) is indicated in A;representative micrographs are shown in B. The scale bar indicates 50μm.

(C, D) Quantification of let-7a (C) and miR-34a (D) expression asdetermined by LNA real-time RT-PCR for the cells lines U2932 (white) andSUDHL4 (black) 48 h after electroporation with Myc-specific (MYC) orscrambled (C=control) siRNA. Expression values were normalized to U6snRNA levels.

(E, F) Proliferation as assessed by [³H] thymidine incorporation ofU2932 (E) and SUDHL4 (F) cells 72 h after electroporation withMyc-specific or scrambled (C=control) siRNA.

(G, H) U2932 (G) and SUDHL4 (H) cells were electroporated with theindicated pre-miRs or scrambled negative control (C) oligonucleotide 72h prior to the quantification of proliferation by [³H] thymidineincorporation.

FIG. 3. MiR-34a directly targets FOXP1 in DLBCL.

(A) U2932 cells were electro-porated with pre-miR34a or a scramblednegative control (C) pre-miR and analyzed with respect to FOXP1expression 48 h later. FOXP1 transcript levels were normalized to GAPDHexpression.

(B) FoxP1 protein levels of the experiment described in A as analyzed byWestern blot. α-Tubulin (α-T) levels are shown to control for equalloading.

(C) Dual luciferase assay of HEK293T cells co-transfected with fireflyluciferase constructs containing the wild-type (WT) or mutant (M)miR-34a target site of the FOXP1 3′-UTR region downstream of theluciferase reporter. Cells were co-transfected with either pre-miR-34aor a negative control (C) scrambled oligonucleotide and the respectiveluciferase construct. Data are represented as relative luciferaseactivity (Luc).

FIG. 4. FOXP1 is a bona fide oncoprotein in DLBCL.

(A, B) FoxP1 expression was analyzed by immunohistochemistry on thetissue microarray described in FIG. 2. The fraction of MALT lymphoma(MALT-L) and gDLBCL cases with high (black), low (grey) and no Mycexpression (white) is indicated in A; representative micrographs areshown in B. The scale bar indicates 50 μm.

(C-F) U2932 (C), SUDHL4 (D), SUDHL6 (E) and SUDHL7 (F) cells wereelectroporated with pre-miR34a, a FoxP1-specific siRNA or the respectivenegative control (C) scrambled oligonucleotides 72 h prior to thequantification of proliferation by [³H] thymidine incorporation.

FIG. 5. Intratumoral delivery of formulated miR-34a blocks DLBCL growthin vivo. Local treatment with mir-34a inhibits the growth of DLBCLxenografts. NOD/SCID mice were inoculated subcutaneously in both flankswith 1×10⁷ U2932 cells. Palpable tumors were locally injected every 3days either with 12.5 μg miR-34a (n=10) or 12.5 μg scrambled negativecontrol (NC) miRNA (n=12) for a period of 15 days.

(A) Tumor volume (mm³) was calculated via caliper measurements every 3days using the formula: (a²×b)/2, where a is the shorter and b thelonger dimension of the tumor. Tumor volume (mm³) is shown as functionof days of treatment with either miR-34a or negative control (NC). Barsrepresent SEM. P values were obtained using Student's t test: ***,P<0.001.

(B) Scatter plot showing the final tumor weights (gr) of the NC andmiR-34a-treated xenografts.

(C) Quantitative real time PCR analysis of miR-34a levels in tumorstreated with either miR-34a or NC miRNA. Expression values werenormalized against the U6 snRNA. P values were obtained using Student'st test: *, P<0.05; ***, P<0.001. Data represent 3 independentexperiments.

FIG. 6. Systemic treatment with formulated miR-34a suppresses growth ofDLBCL in vivo.

Systemic delivery of mir-34a blocks tumor growth. 1×10⁷ U2932 cells wereinoculated subcutaneously in both flanks of NOD/SCID mice. Once tumorsreached a palpable volume the mice were intravenously injected with 20μg of formulated miR-34a (n=7) or PBS (negative control, NC) (n=7) every2 days. The final injection was performed on day 13, 10 minutes beforethe study endpoint.

(A) Tumor volumes (mm³, determined as explained for FIG. 5(A)) weremeasured every 2 days. Bars represent SEM. P values were obtained usingStudent's t test: *, P<0.05; **, P<0.01,***, P<0.001.

(B) Tumor weights (gr) of the NC and miR-34a-treated xenografts.

(C) miR-34a levels were measured by quantitative real time PCR in tumorstreated with either miR-34a or NC miRNA. Expression values werenormalized against the U6 snRNA. Data represent 2 independentexperiments.

DETAILED DESCRIPTION OF THE INVENTION

Real time RT-PCR revealed the microRNA miR-34a to be differentiallyregulated in gastric low grade MALT lymphoma and its transformed highgrade disease counterpart, gastric diffuse large B-cell lymphoma(gDLBCL). miR-34a is negatively regulated by Myc, an oncogeneoverexpressed in gDLBCL. Bioinformatic target prediction combined withfunctional analyses revealed that miR-34a represents a tumor suppressormiRNA in gDLBCL, which acts through post-transcriptional control of itsdirect target FoxP1, a hematopoietic oncoprotein overexpressed ingDLBCL. These findings identify a new mechanism that links the aberrantexpression of Myc and the resulting repression of the tumor suppressormiRNA miR-34a to FoxP1 deregulation in high grade transformation ofgastric B-cell lymphoma.

Related observations have been made in Burkitt lymphoma and in chroniclymphocytic leukemia, two non-gastric mature B-cell neoplasms.

The invention relates to microRNA, such as microRNA-34a, for use in thetreatment of B-cell lymphoma, in particular gastric B-cell lymphoma,non-gastric diffuse large B-cell lymphoma, extranodal diffuse largeB-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules 13 to 25nucleotides long. MicroRNAs are post-transcriptional regulators thatbind to complementary sequences in the 3′ untranslated regions (3′ UTRs)of target messenger RNA transcripts usually resulting in gene silencing.

MicroRNA-34a (miR-34a) is a member of the miR-34 family, which iscomposed of miR-34a, miR-34b, miR-34c-5p and miR-34c-3p. The miR-34agene is located on chromosome 1p36.22. MicroRNAs considered in thepresent invention are miR-34a, miR-34b, miR-34c-5p, miR-34c-3p, miR-15a,miR-23a, miR-26a, miR-150, and let-7a.

MicroRNA-34a has the sequence UGGCAGUGUCUUAGCUGGUUGU (SEQ ID NO:1).

MicroRNA-34b has the sequence CAAUCACUAACUCCACUGCCAU (SEQ ID NO:2).

MicroRNA-34c-5p has the sequence AGGCAGUGUAGUUAGCUGAUUGC (SEQ ID NO:3).

MicroRNA-34c-3p has the sequence AAUCACUAACCACACGGCCAGG (SEQ ID NO:4).

MicroRNA-15a has the sequence UAGCAGCACAUAAUGGUUUGUG (SEQ ID NO:5).

MicroRNA-23a has the sequence AUCACAUUGCCAGGGAUUUCC (SEQ ID NO:6).

MicroRNA-26a has the sequence UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO:7).

MicroRNA-150 has the sequence UCUCCCAACCCUUGUACCAGUG (SEQ ID NO:8).

MicroRNA let-7a has the sequence UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO:9).

Gastric B-cell lymphoma is a non-Hodgkin's lymphoma of the stomach. Inparticular, gastric B-cell lymphoma is high-grade gastric diffuse largeB-cell lymphoma (gDLBCL). Burkitt lymphoma is a further B-cellnon-Hodgkin lymphoma. Chronic lymphocytic leukemia (CLL) is the mostcommon leukemia, and also represents a B-cell lymphoma. The inventioncan also be applied to non-gastric diffuse large B-cell lymphoma andextranodal diffuse large B-cell lymphoma.

The invention further relates to microRNA, such as microRNA-34a, for usein the preparation of a medicament for the treatment of B-cell lymphoma,in particular gastric B-cell lymphoma, non-gastric diffuse large B-celllymphoma, extranodal diffuse large B-cell lymphoma, Burkitt lymphoma andchronic lymphocytic leukemia.

For the administration, the active ingredient is preferably in the formof a pharmaceutical preparation comprising a microRNA as activeingredient in chemically pure form or suitably derivatized, andoptionally a pharmaceutically acceptable carrier and optionallyadjuvants.

The microRNA is used in an amount effective against the disease inhumans. The dosage of the active ingredient depends upon the age,weight, and individual condition of the human being, the individualpharmacokinetic data, and the mode of administration. In the case of anindividual human having a bodyweight of about 70 kg the daily doseadministered of a microRNA is from 0.01 mg/kg bodyweight to 100 mg/kgbodyweight, preferably from 0.1 mg/kg bodyweight to 50 mg/kg bodyweight,more preferably from 1 mg/kg to 20 mg/kg bodyweight administered as asingle dose or as several doses. The microRNA can be used alone or incombinations with other drugs.

MicroRNAs of the invention are applied in unmodified form, preferably asduplexes involving complementary RNA (double-stranded RNA), chemicallymodified in part as 2′-O-methylpurines or 2′-fluoropyrimidines, or asasymmetrical Dicer substrates with a blunt end which includes two DNAbases and a two nucleotide overhang at the 3′ end. Dicer is anendoribonulclease of the RNases III family that cleaves double-strandedRNA into short double stranded RNA fragments and catalyzes formation ofthe RNA-induced silencing complex. Another envisaged derivative ofmicroRNA is double-stranded RNA chemically bound at the 3′ hydroxy groupto cholesterol. Further derivatives considered are those indicated inKim D. H. and Rossi J. J., Nature Reviews Genetics, 2007, 8:173-184.

Pharmaceutical compositions for parenteral administration, such assubcutaneous, intravenous, intrahepatic or intramuscular administration,are especially preferred. The pharmaceutical compositions comprise fromapproximately 1% to approximately 95% active ingredient, preferably fromapproximately 20% to approximately 90% active ingredient.

For parenteral administration preference is given to the use ofsolutions of the microRNA and microRNA derivatives, and also suspensionsor dispersions, especially isotonic aqueous solutions, dispersions orsuspensions which, for example, can be made up shortly before use. Thepharmaceutical compositions may be sterilized and/or may compriseexcipients, for example preservatives, stabilizers, wetting agentsand/or emulsifiers, solubilizers, viscosity-increasing agents, salts forregulating osmotic pressure and/or buffers and are prepared in a mannerknown per se, for example by means of conventional dissolving andlyophilizing processes.

In particular, formulations for parenteral administration may contain,for example, excipients, sterile water or saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, biodegradablelactide polymers, lactide/glycolide copolymers,polyoxyethlene-polyoxypropylene copolymers, ethylene-vinyl acetatecopolymers, cyclodextrins, porphyrin derivatives, polyethyleniminepolymers, lipofectin, atelocollagen, polylysine, nanoparticles,microspheres and liposomes, in particular liposomes formed fromphospholipid bilayers.

Liposomes suitable in the invention are formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors such asdesired liposome size and half-life of liposome in the blood stream.Further considered are liposomes modified so as to avoid clearance bythe mononuclear macrophages and reticuloendothelial systems, for examplehaving opsonization-inhibition moieties bound to the surface of theliposome structures. Opsonization-inhibition moieties are largehydrophilic polymers bound to the liposome membrane, for examplepolyethylene glycol or polypropylene glycol and derivatives thereof,e.g., methoxy derivatives or stearates, or also synthetic polymers suchas polyacrylamide or polyvinyl-pyrrolidone, linear, branched ordendrimeric polyamidoamines, polyacrylic acids, polyalcohols, e.g.polyvinyl alcohols and polyxylitol, and gangliosides. Preferredopsonization-inhibition moieties are polyethylene glycol orpolypropylene glycol and derivatives thereof giving rise to “pegylatedliposomes”, resulting in stable nucleic acid-lipid particles (SNALPs,Kim D. H. and Rossi J. J., Nature Reviews Genetics, 2007, 8:173-184).

Further considered are pharmaceutical compositions for selectivesystemic delivery, for example coupled to antibody fragments, aptamersor packaged into nanoparticles coated with receptor-targeting ligands.

The microRNA can be administered alone or in combination with one ormore other therapeutic agents, possible combination therapy taking theform of fixed combinations of the microRNA and one or more othertherapeutic agents known in the treatment of gastric B-cell lymphoma inhumans, the administration being staggered or given independently of oneanother, or being in the form of a fixed combination.

Possible combination partners considered are cyclophosphamide,hydroxydaunorubicin (doxorubicin, adriamycin), oncovin (vincristine),prednisone/prednisolone, and rituximab.

Pharmaceutical preparations according to the invention are manufacturedby methods known in the art, especially by conventional mixing, coating,granulating, dissolving or lyophilizing.

The invention further relates to a method of treatment of B-celllymphoma, in particular gastric B-cell lymphoma, non-gastric diffuselarge B-cell lymphoma, extranodal diffuse large B-cell lymphoma, Burkittlymphoma and chronic lymphocytic leukemia, comprising administering to apatient in need thereof a therapeutically effective amount of amicroRNA, such as microRNA-34a.

The scientific rationale for the use of a microRNA, such asmicroRNA-34a, in the treatment of gastric B-cell lymphoma and relatedB-cell lymphoma is as follows:

miR-34a is Specifically Down-Regulated in High Grade-Transformed GastricLymphoma

Total RNA isolated from 7-8 cases each of Helicobacter-associatedreactive gastritis, low grade MALT lymphoma and high grade gDLBCL wassubjected to real time RT-PCR in order to determine the differentialexpression patterns of two selected miRNAs, let-7 and miR-34a (FIG. 1 A,B). In conclusion, it was found that high grade transformation ofgastric MALT lymphoma is accompanied by down-regulation of miR-34a.

Myc is Over-Expressed in High Grade Gastric DLBCL and Controls DLBCLProliferation In Vitro

Since Myc is known to negatively regulate miR-34a expression, the Mycexpression status of a set of 37 gDLBCL and 39 low grade MALT lymphomasspotted onto a gastric lymphoma tissue microarray was assessed. Indeed,80% of gDLBCL, but only 20% of low grade lymphomas showed highexpression of Myc (FIG. 2 A, B), indicating that Myc expression may be auseful marker for the differential diagnosis of both disease entities.To assess a possible causal link between Myc expression and miRNAdown-regulation in DLBCL, Myc expression was transiently knocked down intwo DLBCL lines, of which one had the characteristics of the ‘activatedB-cell’ type of DLBCL (‘ABC’; U2932) and the other had typical ‘germinalcenter’ type features (‘GC’, SUDHL4). The transient knock-down of Mycindeed increased expression of both let-7 (FIG. 2C) and miR-34a (FIG.2D) in both cell lines in relation to a scrambled siRNA. Interestingly,the proliferation of both cell lines as determined by [³H] thymidineincorporation was significantly reduced upon siRNA-mediated Myc knockdown (FIG. 2 E, F), indicating that Myc expression drives lymphoma cellproliferation, possibly via down-regulation of tumor suppressive miRNAs.

To determine which of the Myc-repressed miRNAs have tumor suppressiveproperties in DLBCL cell lines in vitro, a panel of six miRNAs that wereconsistently predicted by both the TargetScan and PicTar algorithms totarget known or putative hematopoietic oncogenes such as Bcl6, Ezh2,FoxP1 and Pax5 were analyzed. Synthetic, chemically modifieddouble-stranded precursor molecules of the six miRNAs (so-calledpre-miRs™) were introduced into U2932 and SUDHL4 cells by nucleoporationusing the Amaxa system, either alone or in combination (FIG. 2 G, H).While all miRNAs on this panel had suppressive effects on tumor cellproliferation in relation to an unspecific negative control miRNA, onecandidate, miR-34a, was particularly effective in this respect (FIG. 2G, H). miR-34a is a known tumor suppressor miRNA in prostate and lungcancer and is a lead candidate for miRNA replacement therapy for thetreatment of these malignancies (Wiggins J. F. et al., Cancer Res 2010,70:5923-30). This combined bioinformatic and experimental approachidentified a miRNA with interesting tumor suppressive characteristics inDLBCL in vitro.

miR-34a Targets the Transcription Factor FoxP1 in DLBCL

Bioinformatically predicted targets of miR-34a include the transcriptionfactors Bcl6 and FoxP1, which harbor one and two putative miR-34a seedregions in their 3′ untranslated region (UTR), respectively. As bothhave previously been linked to the pathogenesis of gDLBCL, theirpossible post-transcriptional regulation by miR-34a was tested in theDLBCL cell lines introduced earlier. Quantitative RT-PCR of FoxP1 andBcl6 expression after nucleoporation of U2932 and SUDHL4 cells withpre-miR-34a revealed that FoxP1, but not Bcl6, is a likely direct targetof this miRNA (FIG. 3A). Protein levels of FoxP1 were also stronglyreduced upon introduction of miR-34a into U2932 cells (FIG. 3B). Inorder to measure a direct effect of miR-34a binding to its seed regionsin the foxp1 gene, the wild type sequence of the seed region, or amutant version in which four of the six positions had been mutated, wascloned downstream of a luciferase reporter gene. Co-transfection ofpre-miR-34a with the luciferase expression vector harboring the wildtype seed region, but not the mutant version, blocked reporter geneexpression as assessed by luciferase activity assay (FIG. 3C). Theresults demonstrate that FoxP1, but not Bcl6, is a target of miR-34a inDLBCL cell lines.

The miR-34a Target FoxP1 is a Bona Fide Oncoprotein in DLBCL

As FoxP1 is a direct target of miR-34a, and an indirect target of Myc,it was tested whether FoxP1 is differentially expressed in gDLBCL andlow grade MALT lymphoma. Indeed, a majority of gDLBCL, but very few ofthe low grade lymphoma cases spotted onto the tissue microarray showedreactivity with a FoxP1-specific antibody (FIG. 4 A, B). Interestingly,all FoxP1-positive cases also expressed Myc, irrespective of whetherthey were classified as low grade or high grade lymphomas. On the otherhand, FoxP1 expression did not overlap with expression of Bcl6, which isoften used to distinguish between ‘GC’ and ‘ABC’ type DLBCL: similarproportions of FoxP1-positive cases were Bcl6-positive and -negative. AnsiRNA-mediated knock down of FoxP1 should have similaranti-proliferative effects in DLBCL cell lines as the delivery ofmiR-34a. This was indeed the case: knock down of FoxP1 blocked theproliferation of four ‘GC’ and ‘ABC’ type DLBCL cell lines at similarlevels, and roughly as efficiently as the re-introduction of miR-34a(FIG. 4 C-F). In conclusion, the results suggest that the transcriptionfactor FoxP1 is a direct target of miR-34a in gDLBCL and isoverexpressed in a majority of gDLBCL, but not in low grade lymphomacases, and represents a bona fide oncoprotein in this disease entity.

Intratumoral Delivery of microRNA-34a Abrogates Growth of Diffuse LargeB-Cell Lymphoma In Vivo

Based on a previous study (Wiggins J. F. et al., Cancer Res 2010,70:5923-30) documenting the efficacy of miRNA replacement in preventingtumor growth in mouse models of non-small-cell lung cancer, a possibletherapeutic effect of intratumorally administered miR-34a was examinedin a xenograft model of DLBCL. Severely immunocompromisedNOD/SCID/IL2Rg^(−/−) mice lacking B-, T- and NK cells were inoculatedsubcutaneously with U2932 cells, an ABC type DLBCL cell line with no orvery little miR-34a expression due to promoter hypermethylation. Oncepalpable tumors of ˜50 mm² had formed, the mice received regularintratumoral doses of miR-34a or negative control miRNA formulated inlipid-based transfection reagent at three-day intervals for 15 days.Whereas the tumors in the negative control group grew rapidly in thistime frame, a strong reduction of tumor growth was observed in themiR-34a-treated group (FIG. 5A). The difference in tumor volume betweenthe treatment groups was evident macroscopically, and was reflected insignificantly different tumor weights at the study endpoint (FIG. 5B).Whereas miR-34a expression was hardly detectable in the tumors of thenegative control group, its levels were strongly increased in themiR-34a-treated group (FIG. 5C). The difference in miR-34a expressionwas not due to miRNA accumulating in the interstitial space, but ratherreflected its uptake into the tumor cells as assessed in single cellsuspensions after 24 hours of culturing ex vivo.

Systemic Delivery of microRNA-34a Abrogates Growth of Diffuse LargeB-Cell Lymphoma In Vivo

Based on the promising results of the intratumoral treatments, astrategy to systemically deliver miR-34a or control miRNA was devised inthe U2932 xenograft model. Synthetic miR-34a mimic or control miRNA wasformulated in neutral lipid emulsion and the therapeutic benefit ofregular intravenous injections examined in the U2932 model.NOD/SCID/IL2Rg^(−/−) mice subcutaneously implanted with U2932 cells werestarted on the treatment once their tumors had reached a size of ˜50mm²; each mouse received a total of eight doses and was monitoredclosely with respect to its tumor volume and possible side effects forthe duration of the treatment. None of the treated mice exhibitedadverse symptoms in the course of the treatment, nor did any of the miceincluded in the study develop abnormalities that would have been evidentat necropsy. As with the intratumoral treatment, a strong reduction intumor growth was observed in mice receiving NLE-formulated miR-34a mimiccompared to mice in the negative control group (FIG. 6A). Thedifferences in tumor volume between the treatment groups were againreflected in significantly different tumor weights at the study endpoint(FIG. 6B). The tumors of miR-34a-treated mice exhibited substantiallyhigher levels of miR-34a than control tumors (FIG. 6C); other organssuch as the liver and spleen also differed between the treatment groupsin terms of miR-34a expression levels, but not to the same extent as thetumors themselves. This result can probably be attributed to the factthat the nucleotide sequence of mature miR-34a of mice and humans isidentical, and that the quantitative PCR therefore cannot differentiatebetween the endogenously expressed miRNA and the ectopically deliveredmimic. As the miR-34a baseline expression is higher in liver tissue thanin the xenograft tumors, its accumulation upon systemic administrationis more evident in the tumors. In conclusion, systemic delivery ofmiR-34a to xenograft DLBCL tumors is feasible and efficiently preventstumor growth without producing adverse side effects; the results implythat miRNA replacement is suitable for the treatment of DLBCL patientswith transcriptional or epigenetic silencing of this and related tumorsuppressor miRNA.

The results taken together establish a mechanistic link betweenoverexpression of Myc, the concomitant repression of Myc-regulated miRNAgenes, and the deregulation of FoxP1. Chromosomal translocationsinvolving the MYC locus are reported in the literature for 50-60% ofgDLBCL, but are never detected in gastric low grade lymphomas and rarelyfound in nodal DLBCL. While chromosomal translocations involving the MYClocus are the likely cause for Myc overexpression in gDLBCL, otheralternative mechanisms of Myc deregulation are conceivable. Myc is knownto be post-transcriptionally regulated by miRNAs. Interestingly, severalMyc-regulating miRNAs (including miR-34a and let7) are also targets ofMyc repression, establishing a feedback loop that aggravates andperpetuates the effects of Myc overexpression and may thus contribute tocancer progression. Several lines of evidence argue that miR-34a is astrong tumor suppressor miRNA in solid cancers. It is located onchromosome 1p36.22 in a region that has previously been associated withvarious malignancies, including lung cancer; it is transcriptionallyinduced by the tumor suppressor p53 and its overexpression inhibitsgrowth of various cancer types in vitro. The first proof of principlefor the success of miR-34a “replacement therapy” in cancer treatment wasrecently reported using a preclinical model of non-small cell lungcancer; the local and systemic delivery of chemically synthesizedmiR-34a was achieved in this model by formulation with a lipid-baseddelivery reagent (Wiggins J. F. et al., Cancer Res 2010, 70:5923-30).

It is demonstrated here for the first time that miR-34a has tumorsuppressive properties in a hematopoietic malignancy. It isdown-regulated in the malignant compared to the benign form of gastriclymphoma and its expression is directly regulated by Myc, which is shownto possess oncogenic properties in various DLBCL cell lines. EctopicmiR-34a re-expression prevents DLBCL growth in vitro; its intratumoralor systemic delivery prevents tumor growth in a xenograft model.Finally, it is demonstrated that miR-34a targets a suspectedhematopoietic oncogene in gastric lymphoma, FoxP1, which isover-expressed in the malignant form of gastric lymphoma and whichpossesses bona fide oncogenic properties in DLBCL. Thepost-transcriptional regulation of FoxP1 by miR-34a further provides aplausible explanation for the conundrum that FoxP1 is highly expressedin many lymphomas not harboring the rare chromosomal translocationt(3;14)(p13;q32), which juxtaposes the FOXP1 and IGH gene loci incertain non-gastric MALT lymphomas and extranodal DLBCL.

MiR-34 replacement therapy should therefore be considered for thetreatment of patients with miR-34a-negative, FoxP1-overexpressinghematopoietic malignancies such as gastric DLBCL. The beneficialtherapeutic effects of miR-34a are due to the strong tumor suppressiveproperties of this miRNA, which are exerted via its oncogene targetFoxP1.

Patient Material and DLBCL Cell Lines

For miRNA expression analysis of archived patient material, consecutivecases of H. pylori-positive gastritis, of H. pylori-positive gastric lowgrade MALT lymphoma, and of gastric high grade MALT lymphoma were drawnfrom the surgical pathology files of the Institute of Pathology at theCantonal Hospital St. Gallen, Switzerland. All data were blinded toguarantee patients' protection. All procedures were in agreement withthe guidelines for use of human material in research issued by theparticipating Institutions' Ethics Committees.

RNA Extraction and Locked Nucleic Acid Real Time PCR for microRNAQuantification

Total RNA was extracted from three 20 μm slices per sample of formalinfixed, paraffin embedded material using the RecoverAll total RNAIsolation kit (Ambion, Streetsville, Canada). Total RNA concentrationswere measured using an ND-1000 spectrophotometer (NanoDropTechnologies). RNA integrity was confirmed on an Agilent 2100Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA). Theexpression of mature miRNAs was analysed using the miRCURY lockednucleic acid (LNA) microRNA PCR system following the manufacturer'sinstructions (Exiqon, Vedbaek, Denmark). 10 ng of total RNA wassubjected to cDNA synthesis using either miRNA- or U6 snRNA-specificprimers. The cDNA template was diluted 1:10 and real time PCR reactionswere performed following the manufactures' recommendations (LightCycler;Roche, Basel, CH). Calculations of miRNA expression levels wereperformed using the comparative ΔΔ_(t) method and normalized against U6snRNA levels.

Immunohistochemical Staining and Western Blotting

The gastric lymphoma tissue microarray used in this study wasconstructed as described (Bernasconi B. et al., Hum Pathol 2008,39:536-42) and included a total of 76 specimens, comprising 39 cases ofgastric low grade MALT lymphomas and 37 cases of gastric DLBCL. Thefollowing primary antibodies were used: anti-MYC (N-262; Santa CruzBiotechnology, Santa Cruz, Calif., USA) and anti-FOXP1 (ICI2; Abcam,Cambridge, Mass., USA). MYC and FOXP1 levels were assessed by countingthe number of positively staining tumour cells and graded using thefollowing expression scale: a negligible level of staining of 0-10% wasrecorded as negative, while low expression was between 10-60% and highexpression was recorded when 60-100% tumour cells stained positive.Protein extracts were made using 2× Laemmli sample buffer (4% SDS, 20%glycerol, 120 mM Tris pH 6.8). Proteins were separated bySDS/polyacrylamide gel electrophoresis and transferred ontonitrocellulose membranes. Membranes were probed with antibodies againstFOXP1 (JC12; Abcam) or against α-tubulin (Santa Cruz Biotechnology) tocontrol for equivalent gel loading.

Cell Lines, Transfections and Luciferase Reporter Assays

On-target plus smartpool siRNAs for MYC, FOXP1 and a scrambled negativecontrol was purchased from Dharmacon (Thermo Scientific, Lafayette,Colo., USA). Precursor microRNA oligonucleotides (pre-miR-let-7a,pre-miR-34a, pre-miR-23a, pre-miR-26a, pre-miR-150 and pre-miR-15) andscrambled negative control oligonucleotides were purchased from Ambion.For the purpose of siRNA or miRNA introduction into DLBCL cells, 1×10⁶cells were nucleoporated using an Amaxa Nucleoporator (Gaithersburg,Md., USA) with the specified amount of pre-miR miRNA precursor or siRNA.After 48 h, cells were harvested for RNA and protein analysis. After 72h, tumor cell proliferation was quantified by [³H]-thymidineincorporation assay as previously described (Craig V. J. et al., Blood2010, 115:581-91). The pmirGLO Dual-Luciferase miRNA Target ExpressionVector was purchased from Promega (Madison, Wis., USA). HEK293T cellswere seeded into 21-well plates at 1×10⁵ cells/well 24 h beforetransfection. 1 μg reporter plasmid containing the FOXP1 3′UTR or itsmutants and 30 nM mir-34a precursor molecules were contransfected intoeach well using the Fugene 6 transfection reagent (Roche) intriplicates. Luciferase assays were performed 24 h after transfectionusing the Dual-Luciferase Reporter Assay System (Promega) with aSpectramax M5 reader (Molecular Devices, Sunnyvale, Calif., USA).

1. A microRNA for use in the treatment of B-cell lymphoma.
 2. A microRNAfor use in the treatment of gastric B-cell lymphoma, non-gastric diffuselarge B-cell lymphoma, extranodal diffuse large B-cell lymphoma, Burkittlymphoma and chronic lymphocytic leukemia according to claim
 1. 3. AmicroRNA for use in the treatment of high-grade gastric diffuse largeB-cell lymphoma according to claim
 1. 4. The microRNA for use in thetreatment of B-cell lymphoma according to claim 1, 2 or 3 selected fromthe group consisting of miR-34a, miR-34b, miR-34c-5p, miR-34c-3p,miR-15a, miR-23a, miR-26a, miR-150, and let-7a.
 5. MicroRNA-34a for usein the treatment of B-cell lymphoma according to claim 1, 2 or
 3. 6. Acombination of a microRNA and another active ingredient selected fromcyclophosphamide, hydroxydaunorubicin, vincristine,prednisone/prednisolone, rituximab, and combinations thereof, for use inthe treatment of B-cell lymphoma.
 7. The combination of claim 6 for usein the treatment of B-cell lymphoma wherein microRNA is microRNA-34a. 8.The combination according to claim 6 or 7 for use in the treatment ofhigh-grade gastric diffuse large B-cell lymphoma.
 9. A microRNA for usein the preparation of a medicament for the treatment of B-cell lymphoma.10. MicroRNA-34a for use in the preparation of a medicament for thetreatment of B-cell lymphoma according to claim 9
 11. A method oftreatment of B-cell lymphoma comprising administering to a patient inneed thereof a therapeutically effective amount of a microRNA.
 12. Themethod of claim 11 wherein the microRNA is microRNA-34a.